昆虫细胞凋亡蛋白酶caspase-1的表达纯化及活性分析

昆虫细胞凋亡蛋白酶caspase-1的表达纯化及活性分析 Expression and Activity Analysis of Caspase-1 from Insect Cells 曾宪东 ZENG Xiandong1 1 2 first-author 宋建华 SONG Jianhua1 1 梁昌镛 LIANG Changyong1 1 陈新文 CHEN Xinwen1 1 中国科学院武汉病毒研究所/病毒学国家重点实验室 中国科学院武汉病毒研究所/病毒学国家重点实验室 Wuhan Institute of Virology,Chinese Academy of Sciences/State Key Laboratory of Virology Wuhan Institute of Virology,Chinese Academy of Sciences/State Key Laboratory of Virology 中国科学院研究生院 中国科学院研究生院 Graduate University of Chinese Academy of Sciences Graduate University of Chinese Academy of Sciences 依赖于天冬氨酸的半胱氨酸蛋白酶（caspase）在细胞凋亡途径中起着不可替代的关键酶作用.本研究采用原核表达载体pET32a表达和纯化了家蚕细胞Bm-caspase-1及粉纹夜蛾细胞Tni-caspase-1蛋白酶,序列分析表明二者具有相同的内部剪切识别序列,且酶活性位点均为QACQ↓G.纯化获得大量可溶性蛋白酶,Western-blotting分析表明Bm-caspase-1在大肠杆菌中已自我剪切活化,而Tni-caspase-1没有自我剪切,无酶活性;活性Bm-caspase-1蛋白酶对caspase-3特异性底物Ac-DEVD-AMC的降解酶活性可被Z-VAD-FMK呈剂量依赖性抑制,半抑制浓度约20nmol/L.此外,活性Bm-caspase-1蛋白酶能够转活化无活性的Tni-caspase-1蛋白酶原.结果揭示了昆虫细胞caspase同样具有与哺乳动物细胞caspase相似的底物降解活性并可相互剪切激活,为进一步研究昆虫细胞凋亡的分子途径奠定了基础. Caspase,aspartate-specific cysteine proteases,play an irreplaceable role in apoptosis pathways as key proteinases. Two insect caspases,Bm-caspase-1 from Bombyx mori （domestic silkworm） and Tni-caspase-1 from Trichoplusia ni,were expressed and purificated in E. coli with the prokaryotic expression vector pET32a. There are the same active sites of pentapeptide QACQG in their amino acid sequences. Western blotting analysis indicated that Bm-caspase-1 could be activated by self-cleavage in E.coli,but not Tni-caspase-1. The proteinase activity of purificated Bm-caspase-1 cleaving Ac-DEVD-AFC,an specific substrate for human caspase-3,could be inhibited by Z-VAD-FMK in a dose-dependent manner and the IC50 of Z-VAD-FMK was about 20 nmol/L for 1 mg/L Bm-caspase-1. In addition,active Bm-caspase-1 activated the non-activated proenzyme of purificated Tni-caspase-1 in vitro. These results indicated that the insect caspases are similar to the mammalian caspases in terms of substrate-cleaving specificity and the activating manner by self-cleavage and trans-cleavage. Bm-caspase-1 Tni-caspase-1 表达 纯化 活性分析 Bm-caspase-1 Tni-caspase-1 expression purification activity analysis 10.14188/j.1671-8836.2010.01.003 Q78 国家基础研究重点项目（2003CB1140）;国家自然科学基金（30325002,30670077）;荷兰皇家艺术和科学院资助项目（KNAW,Program Strategic Scientific Alliances Project 04-PSA-BD-0） 2010-01-01 2021-04-01 1 56